Abstract

The excitation of the renal sympathetic nervous system plays an important role in the development of ischemic acute kidney injury (AKI) in rats. We have reported that intravenous treatment with GABA has preventive effects on ischemia/reperfusion (I/R)-induced renal dysfunction with histological damage in rats. However, detailed mechanisms of the action of GABA on the renal injury were still unknown. Therefore, in the present study, we aimed to clarify the detailed mechanisms of GABA in ischemic AKI in rats. Ischemic AKI was induced by clamping the left renal artery and vein for 45 min. Thereafter, the kidney was reperfused to produce I/R-induced injury. Intravenous or intracerebroventricular treatment with 3-[[[(3,4-dichlorophenyl)methyl]amino]propyl] diethoxymethyl) phosphinic acid (CGP52432), a GABAB receptor antagonist, abolished the suppressive effects of intravenously applied GABA on enhanced renal sympathetic nerve activity during ischemia, leading to the elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with GABA or intravenous treatment with baclofen, a selective GABAB receptor agonist, prevented I/R-induced renal injury equivalent to intravenous treatment with GABA. However, intravenous treatment with bicuculline, a GABAA receptor antagonist, failed to affect the preventive effects of GABA on ischemic AKI. Therefore, we demonstrated the novel finding that the preventive effect of GABA on ischemic AKI through the suppression of enhanced renal sympathetic nerve activity induced by renal ischemia is presumably mediated via GABAB receptor stimulation in the central nervous system rather than peripheral GABAB receptor.

Introduction

Acute kidney injury (AKI) is encountered commonly in the hospital and is associated with a high rate of mortality (Lattanzio and Kopyt, 2009). Ischemia, followed by reperfusion, is one of the major causes of AKI (Thadhani et al., 1996). Reperfusion of previously ischemic renal tissue initiates complex cellular events that result in the injury and eventual death of renal cells due to a combination of apoptosis and necrosis (Lieberthal and Levine, 1996). It has been reported that several causal factors (ATP depletion, reactive oxygen species, phospholipase activation, neutrophil infiltration, vasoactive peptides, etc.) contribute to the pathogenesis of this renal damage (Edelstein et al., 1997; Lien et al., 2003). However, the mechanisms underlying ischemia/reperfusion (I/R)-induced renal injury are not fully understood. We found that enhancement of renal sympathetic nerve activity (RSNA) and its consequent effect on norepinephrine (NE) overflow from nerve terminals are considered to be involved in the development of I/R-induced AKI and that RSNA is augmented significantly during renal ischemia in the rats (Fujii et al., 2003; Kurata et al., 2006). In addition, we noted that ischemic AKI is ameliorated by renal denervation or ganglionic blockade and that the effect is accompanied by the suppression of elevated NE levels in the renal vein after reperfusion (Fujii et al., 2003).

GABA, an inhibitory neurotransmitter in the central nervous system (CNS), also is found in peripheral tissues (Gladkevich et al., 2006). GABA is known to suppress electrical renal nerve stimulation-induced NE release from isolated rat kidney without affecting basal release (Fujimura et al., 1999). These findings indicate that GABA can modulate peripheral neurotransmission as well as that in the CNS. It has been reported that GABA has renoprotective effects against glycerol-induced AKI (Kim et al., 2004). We recently reported that preischemic treatment with GABA exerted a suppressive effect on the enhancement of RSNA and caused a consequent elevation of NE levels in the renal vein observed in ischemic AKI rats, suggesting that GABA has renoprotective effects on I/R-induced renal injury (Kobuchi et al., 2009). However, a previous report lacks more precise mechanisms of beneficial action of GABA on AKI and/or I/R-induced renal injury, especially in the subtype of receptors and the site of action of GABA.

Therefore, we investigated the effects of intravenous treatment with bicuculline, a GABAA receptor antagonist, and intravenous treatment with 3-[[[(3,4-dichlorophenyl)methyl]amino]propyl] diethoxymethyl) phosphinic acid (CGP52432), a GABAB receptor antagonist, on the renoprotective effects of GABA to clarify the receptor subtypes responsible for the effects. Furthermore, we examined the effects of intracerebroventricular treatment with CGP52432 on the renoprotective effects of GABA to clarify the site of action. The effects of intravenous treatment with baclofen, a GABAB receptor agonist, and intracerebroventricular treatment with GABA on ischemic AKI also were examined.

Materials and Methods

Animals and Experimental Design.

Male Sprague-Dawley rats (10 weeks of age; Japan SLC, Shizuoka, Japan) were used. The animals were housed in a light-controlled room with a 12-h light/dark cycle and were allowed ad libitum access to food and water. Experimental protocols and animal care methods in the experiments were approved by the Experimental Animal Committee of the Osaka University of Pharmaceutical Sciences (Osaka, Japan). Two weeks before the study (at 8 weeks of age), the right kidney was removed through a small flank incision under pentobarbital anesthesia (40 mg/kg i.p.). After a 2-week recovery period, uninephrectomized rats were divided into sham-operated control, vehicle-treated ischemic AKI, and drug-treated ischemic AKI groups. To induce ischemic AKI, the left kidneys of the rats anesthetized with pentobarbital (50 mg/kg i.p.) were exposed through a small flank incision, and the left renal artery and vein were occluded with a nontraumatic clamp for 45 min. At the end of the ischemic period, the clamp was released to allow reperfusion. Each drug used in this study or vehicle (0.9% saline) was administered into the left external jugular vein (1 ml/kg i.v.) or the right lateral cerebral ventricle (3 μl/rat i.c.v.). Intracerebroventricular injection was performed by a 30-gauge stainless steel cannula implanted into the right lateral cerebral ventricle (stereotaxic coordinates, 0.9–1.0 mm posterior to bregma, 1.4–1.6 mm lateral to midline, 3.2–3.3 mm ventral to dura), as described by Paxions and Watson (1998). The intracerebroventricular position of the cannula was confirmed by the staining of all four ventricles after the injection of 5 μl of pontamine sky blue at the end of the experiment. Drugs or vehicle except bicuculline and CGP52432 were injected 5 min before the start of ischemia. Bicuculline or CGP52432 was given at 10 min before ischemia when examining the effects of these drugs on renal protection by GABA. In sham-operated control rats, the left kidney was treated the same as mentioned above except clamping. The animals exposed to 45 min of ischemia were housed in metabolic cages at 24 h after reperfusion, and 5-h urine samples were collected. At the end of urine collection, blood samples were drawn from the thoracic aorta, and then the left kidneys were excised under pentobarbital anesthesia (50 mg/kg i.p.). The plasma was separated by centrifugation and used for the measurement of renal function as mentioned below. The kidneys were used for light microscopic observation as mentioned for histological studies.

Renal Nerve Recording.

For the measurement of RSNA, uninephrectomized rats were anesthetized with pentobarbital (50 mg/kg i.p.). Surgical preparation of the animals and basic experimental techniques were identical to those described previously (Shokoji et al., 2003). RSNA was recorded from the left renal nerve branch before and during ischemia. The nerve was isolated near the aortic-renal arterial junction through a left flank incision and placed on a Teflon-coated stainless steel bipolar electrode. The renal nerve and electrode were covered with silicone rubber. The renal nerve discharge was amplified using a differential amplifier (AVB-11A; Nihon Kohden, Osaka, Japan) with a band-pass filter (low frequency, 50 Hz; high frequency, 1 kHz). The amplified and filtered signal was visualized on a dual-beam oscilloscope (VC-11; Nihon Kohden) and monitored by an audio speaker. The output from the amplifier was integrated by an integrator (EI601G; Nihon Kohden) with 1-s resetting. The output from the integrator was recorded and analyzed with PowerLab (ML750; ADInstruments Pty Ltd., Castle Hill, Australia). For the quantification of RSNA, the height of the integrated nerve discharge was measured for 20 s in each experiment. The changes in nerve activity were expressed as percentages of control resting spontaneous nerve activity. Electrical signals of renal neural activity were recorded directly for the evaluation of changes in RSNA during the 45-min ischemic period.

Parameters of Renal Function.

Blood urea nitrogen (BUN) and creatinine levels in plasma or urine were determined using a commercial assay kit, the BUN-Test-Wako and Creatinine-Test-Wako (Wako Pure Chemicals, Osaka, Japan), respectively. Creatinine clearance (Ccr, ml/min/kg) was calculated from the formula Ccr = Ucr × UF/Pcr, where Ucr and Pcr are creatinine concentration in urine and plasma, respectively, and UF is urine flow.

Histological Studies.

Excised left kidneys were processed for light microscopic observation, according to standard procedures. The kidneys then were preserved in phosphate-buffered 10% formalin, after which the kidneys were chopped into small pieces, embedded in paraffin wax, cut at 4 μm, and stained with hematoxylin and eosin. Histological changes were analyzed for tubular necrosis, proteinaceous casts, and medullary congestion, as described by Caramelo et al. (1996). Tubular necrosis and proteinaceous casts were graded as follows: no damage (0), mild (1; unicellular, patchy isolated damage), moderate (2; damage <25%), severe (3; damage between 25 and 50%), and very severe (4; >50% damage). The degree of medullary congestion was defined as follows: no congestion (0), mild (1; vascular congestion with identification of erythrocytes by magnification, 400×), moderate (2; vascular congestion with identification of erythrocytes by magnification, 200×), severe (3; vascular congestion with identification of erythrocytes by magnification, 100×), and very severe (4; vascular congestion with identification of erythrocytes by magnification, 40×). The scoring of the histological data was performed by independent observers in a double-blind manner.

Drugs.

GABA and (−)-bicuculline methiodide was purchased from Sigma-Aldrich (St. Louis, MO). CGP52432 and baclofen were purchased from Tocris Bioscience (Ellisville, MO). These drugs were dissolved in saline (0.9%). Other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemicals.

Statistical Analysis.

All of the values were expressed as means ± S.E.M. Relevant data were processed by InStat (GraphPad Software Inc., San Diego, CA). Nerve recording studies were analyzed by one-way repeated measures analysis of variance followed by Dunnett's multiple range test for within-group data. For among-group data, we used two-way repeated measures analysis of variance followed by Fisher's protected least significant difference comparison tests in nerve recording, renal function, or NE concentration studies. Histological data were analyzed using a Kruskal-Wallis nonparametric test combined with a Steel-type multiple comparison test. For all of the comparisons, differences were considered significant at P < 0.05.

Results

Effects of Intravenous Treatment with Bicuculline or CGP52432 on GABA-Induced Improvement against Ischemic AKI.

Histological examination revealed severe lesions in the kidneys of vehicle-treated AKI rats at 29 h after reperfusion. These changes were characterized by proteinaceous casts in tubuli in the inner zones of the medulla (Fig. 3B), medullary congestion, and hemorrhage in the outer zone inner stripes of the medulla (Fig. 3G), and tubular necrosis in the outer zone outer stripes of the medulla (Fig. 3L) in a comparison with kidneys from sham-operated rats (Fig. 3, A, F, and K). Intravenous injection of GABA to ischemic AKI rats significantly attenuated the development of all of these lesions (Fig. 3, C, H, and M; Table 1). Treatment with CGP52432 (Fig. 3, E, J, and O) at the higher dose (100 nmol/kg) negated the above improvement induced by GABA. However, intravenous treatment with bicuculline at the higher dose (10 μmol/kg) failed to affect the GABA-induced action (Fig. 3, D, I, and N).

Effects of Intracerebroventricular Treatment with CGP52432 on GABA-Induced Improvement against Ischemic AKI.

As shown in Fig. 4, the suppressing effect of GABA (50 μmol/kg) on RSNA was attenuated partially by the lower dose of intracerebroventricularly applied CGP52432 (0.1 nmol/kg), and the higher dose of CGP52432 (1 nmol/kg) almost abolished the effect of GABA.

As shown in Fig. 5, the GABA-induced improvement of renal dysfunction was attenuated partially by intracerebroventricular treatment with CGP52432 at the lower dose (0.1 nmol/kg), whereas it almost was abolished by treatment with the higher dose (1 nmol/kg).

Effects of Intravenous Treatment with Baclofen or Intracerebroventricular Treatment with GABA on Ischemic AKI.

The enhanced RSNA during the ischemic period was dose-dependently and markedly suppressed by intravenous treatment with baclofen (0.2 and 1 μmol/kg), a selective GABAB receptor agonist, or intracerebroventricular treatment with GABA (0.1 and 0.5 μmol/kg) (Fig. 6).

As shown in Table 2, intravenous treatment with baclofen (0.2 and 1 μmol/kg) significantly prevented I/R-induced renal dysfunction. The effectiveness of the higher dose of baclofen (1 μmol/kg) was equivalent to that of GABA (50 μmol/kg).

Effects of intravenous treatment with baclofen or intracerebroventricular treatment with GABA on BUN, Pcr, Ccr, and UF at 29 h after reperfusion

Values represent the means ± S.E.M. (n = 6).

As shown in Table 2, intracerebroventricular treatment with GABA (0.1 and 0.5 μmol/kg) dose-dependently attenuated I/R-induced renal dysfunction. The effectiveness of intracerebroventricular treatment with GABA (0.5 μmol/kg) was similar to that of intravenous treatment with GABA (50 μmol/kg).

Discussion

The renal sympathetic nerve system and circulating catecholamines are considered to be involved in the pathogenesis of AKI because pharmacological blockade of the sympathetic nervous system exerts an efficient protective effect on AKI (Kurata et al., 2006; Sugiura et al., 2008). We recently found that intravenous treatment with GABA (10 and 50 μmol/kg) in the ischemic AKI rats efficiently suppressed the enhancement of RSNA during the ischemic period and increased NE overflows after reperfusion (Kobuchi et al., 2009). In the present study, we investigated the more precise mechanisms of the renoprotective effects of GABA on AKI, especially on the subtype of receptors and the site of action of GABA.

The receptor subtypes of GABA receptors are well known as GABAA and GABAB receptors. The GABAA receptor is coupled to the ligand-gated chloride ion channel, whereas the GABAB receptor is coupled to G proteins (Macdonald and Olsen, 1994; Kerr and Ong, 1995). The GABAA receptors are distributed widely within the mammalian CNS and exhibit a differential topographical distribution (Palacios et al., 1981). GABA also activates metabotropic GABAB receptors, which are distributed widely within the CNS and also in peripheral autonomic terminals (Bowery et al., 1981). In the present study, we evaluated the receptor subtype involved in the suppressive effects of GABA on I/R-induced enhancement of RSNA and functional and histological renal injury in rats. Results clearly indicated that the suppressive effects of GABA on I/R-induced enhancement of RSNA and functional and histological renal injury were abolished by intravenous treatment with CGP52432, a GABAB receptor antagonist, whereas bicuculline, a GABAA receptor antagonist, failed to affect the suppressive effects of GABA. In addition, intravenous treatment with baclofen, a GABAB receptor agonist, prevented I/R-induced renal injury by suppressing enhanced RSNA. These findings suggest that activation of GABAB receptor, but not GABAA receptor, is responsible for protection against the development of I/R-induced renal dysfunction.

It has been reported recently that the concentration of GABA in the cerebrospinal fluid is increased by intravenous infusion with GABA itself (Al-Awadi et al., 2006). This finding indicates that intravenous injection of GABA may suppress enhanced RSNA during the ischemic period by acting on the CNS. In the present study, we found that intracerebroventricular treatment with CGP52432 almost abolished the suppressive effects of GABA on I/R-induced enhancement of RSNA and renal injury. Furthermore, we have demonstrated that intracerebroventricular treatment with GABA (0.1 and 0.5 μmol/kg) prevented I/R-induced renal injury by suppressing enhanced RSNA. However, intravenous treatment with the same dose of GABA (0.5 μmol/kg) produced no significant influence on I/R-induced renal injury (S. Kobuchi, unpublished observations). These results suggest that GABA exerts its preventive action against postischemic AKI through inhibitory effects on the central sympathetic outflow.

The paraventricular nucleus and the rostral ventrolateral medulla are known to be responsible for controlling the sympathetic outflow (Coote, 2005; Tjen-A-Looi et al., 2009). The microinjection of GABA or GABA receptor agonist into the rostral ventrolateral medulla or paraventricular nucleus decreased blood pressure by inhibiting sympathetic nerve activity (Avanzino et al., 1994; Li and Pan, 2007). Durgam et al. (1999) reported that the microinjection of the GABA receptor agonist into the nucleus of the solitary tract, which is known to be central regulator for baroreflex, increased blood pressure by enhancing the sympathetic outflow. Therefore, suppression of RSNA by GABA seems to be mediated by the rostral ventrolateral medulla and/or paraventricular nucleus rather than the nucleus of the solitary tract.

Several mechanisms underlying the GABA-induced suppressive action on the peripheral sympathetic nervous system have been proposed, including ganglionic blockade and/or inhibition of transmitter release from nerve terminals (Fujimura et al., 1999; Hayakawa et al., 2002; Kimura et al., 2002). In the present study, we did not examine whether GABA can suppress the NE overflow from the peripheral sympathetic nerves by using isolated tissues. In the peripheral sympathetic nerves, systemically applied GABA may prevent I/R-induced renal injury by inhibition of NE release from nerve terminals in the AKI rats, even after elimination of the effect of GABA by intracerebroventricularly applied CGP52432. However, the present study revealed that systemically applied GABA failed to prevent I/R-induced renal injury when treated with CGP52432 intracerebroventricularly. Therefore, these findings suggest that the renoprotective effect of GABA appears to be much more dependent on the CNS than the peripheral sympathetic nerves.

In conclusion, GABA suppressed the enhanced RSNA during ischemia and increased NE overflow after I/R by the activation of the GABAB receptor, but not by that of GABAA receptor, especially on the CNS rather than on the peripheral nerves in the sympathetic nervous system. These inhibitory effects are presumably responsible for renoprotection against I/R-induced renal injury.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Kobuchi, Ayajiki, and Matsumura.

Footnotes

Received January 28, 2011.

Accepted May 31, 2011.

This study was supported in part by a “High Technology Research Center” Project for Private Universities matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology [2002–2006 and 2007–2009].